Symbiotic Escherichia coli strains can better colonize host stinkbugs and outcompete natural symbiotic bacteria, but confer less fitness benefit.

The stinkbug Plautia stali harbors essential gut symbiotic bacteria of the genus Pantoea, whose natural strains differ in cultivability and host benefits. Using this system, we evaluated how laboratory-evolved and genetically engineered symbiotic Escherichia coli strains compete against native Pantoea symbionts and how they influence host fitness. In single infection assays, the native uncultivable symbiont Sym A conferred the highest host performance, whereas the evolved (CmL05G13) and artificial (ΔcyaA) symbiotic E. coli strains supported host survival at levels comparable to cultivable Pantoea symbionts (Sym C-F). In competitive co-infection assays, the symbiotic E. coli strains generally showed unexpectedly strong colonization ability. CmL05G13 outcompeted all the cultivable symbionts Sym C-F and even displaced the native uncultivable symbiont Sym A, whereas ΔcyaA and the nonsymbiotic control E. coli ΔintS were dominated by Sym A at the adult stage. Despite their superior infection competitiveness, the symbiotic E. coli strains provided limited reproductive benefits, behaving as "cheater-like" associates. They were able to invade and dominate the symbiotic organ but failed to match the fitness contributions of native symbionts. These results demonstrate that the experimentally evolved E. coli can rapidly acquire strong colonization ability, surpassing that of the natural symbionts that have coevolved with P. stali in nature. At the same time, the mismatch between infection success and host fitness benefits highlights potential evolutionary conflicts and provides an experimental model for studying the dynamics of cheating, mutualism, and symbiont replacement in vertically transmitted symbioses.IMPORTANCEUnderstanding how novel symbionts invade and displace long-term mutualists is central to the evolution of symbiosis. This study demonstrates that Escherichia coli, originally a nonsymbiotic bacterium, can rapidly evolve potent colonization ability and even outcompete native Pantoea symbionts of the stinkbug Plautia stali. Meanwhile, these competitive E. coli strains confer markedly lower reproductive benefits compared with the native symbionts that have developed an intimate mutualistic association with the host P. stali over evolutionary time, revealing a striking decoupling between infection success and host fitness. This finding highlights the potential for cheater-like microbes to invade vertically transmitted symbioses and destabilize coevolved partnerships. By combining experimental evolution, controlled co-infections, and quantitative analyses, the P. stali-E. coli experimental symbiotic system provides a powerful model for studying the mechanisms and evolutionary dynamics of mutualism, cheating, and symbiont replacement.